Increasing Spectrum in Antimicrobial Resistance of Shigella Isolates in Bangladesh: Resistance to Azithromycin and Ceftriaxone and Decreased Susceptibility to Ciprofloxacin

Antimicrobial resistance of Shigella isolates in Bangladesh, during 2001-2002, was studied and compared with that of 1991-1992 to identify the changes in resistance patterns and trends. A significant increase in resistance to trimethoprim-sulphamethoxazole (from 52% to 72%, p<0.01) and nalidixic acid (from 19% to 51%, p<0.01) was detected. High, but unchanged, resistance to tetracycline, ampicillin, and chloramphenicol, low resistance to mecillinam (resistance 3%, intermediate 3%), and to emergence of resistance to azithromycin (resistance 16%, intermediate 62%) and ceftriaxone/cefixime (2%) were detected in 2001-2002. Of 266 recent isolates, 63% were resistant to ≥3 anti-Shigella drugs (multidrug-resistant [MDR]) compared to 52% of 369 strains (p<0.007) in 1991-1992. Of 154 isolates tested by E-test in 2001-2002, 71% were nalidixic acid-resistant (minimum inhibitory concentration [MIC] ≥32 μg/mL) and had 10-fold higher MIC90 (0.25 μg/mL) to ciprofloxacin than that of nalidixic acid-susceptible strains exhibiting decreased ciprofloxacin susceptibility, which were detected as ciprofloxacin-susceptible and nalidixic acid-resistant by the disc-diffusion method. These strains were frequently associated with MDR traits. High modal MICs were observed to azithromycin (MIC 6 μg/mL) and nalidixic acid (MIC 128 μg/mL) and low to ceftriaxone (MIC 0.023 μg/mL). Conjugative R-plasmids-encoded extended-spectrum ß-lactamase was responsible for resistance to ceftriaxone/cefixime. The growing antimicrobial resistance of Shigella is worrying and mandates monitoring of resistance. Pivmecillinam or ciprofloxacin might be considered for treating shigellosis with caution.


INTRODUCTION
Shigellosis is one of the significant causes of diarrhoeal diseases in the developing world. Worldwide, an estimated 165 million cases and 1.1 million deaths (mostly in developing countries) occur annually (1)(2)(3)(4). Antimicrobial therapy has been recommended for patients with shigellosis because it can limit the clinical course of illness and reduce the risk of complications and the duration of faecal excretion of the causative organism, reducing the spread of infection (2)(3)(4)(5). The therapy also improves the growth and nutritional status of affected children, especially in developing countries (5). A major problem, however, is the increasing resistance of Shigella spp. to useful antimicrobial agents (3,(6)(7)(8)(9)(10)(11). 11), resulting in difficulties in the selection of empirical therapy. However, newer antimicrobial agents, such as mecillinam, ciprofloxacin, ceftriaxone, cefixime, and azithromycin, were found to be effective in treating multidrug-resistant Shigella-associated infections (6,(12)(13)(14). Recently, Shigella strains resistant to ciprofloxacin have emerged in Bangladesh and India (8,(15)(16).
Currently, we do not have sufficient information on the susceptibility patterns of Shigella isolates to many useful antimicrobial agents, such as mecillinam, ciprofloxacin, levofloxacin, azithromycin, ceftriaxone, and cefixime. Given the impact of suboptimal use of antimicrobial agents in Bangladesh and the ability of Shigella to develop resistance after the introduction of new antimicrobial agents for treatment, it is not unlikely that antimicrobial resistance patterns in Bangladesh have changed since they were reported in the 1990s (11). Thus, the continuing changing patterns of resistance of Shigella isolates indicate the need for monitoring antimicrobial susceptibility. We, therefore, investigated the trends in antimicrobial resistance of Shigella isolates and compared these with previous data to suggest timely recommendations for empirical antimicrobial therapy, if needed.

Clinical samples and bacterial strains
The sources of faecal samples were diarrhoeal patients who attended the Dhaka Hospital of ICDDR,B and whose clinical conditions required stool cultures in a microbiology laboratory as suggested by physicians during the study period. All relevant laboratory data were archived. We analyzed resistance to useful antimicrobial agents of 369 (8%) of 4,597 Shigella strains isolated in the Clinical Laboratory of ICDDR,B, which were collected monthly (32-33 strains per month) during 1991-1992. To compare data on antimicrobial resistance with those of 1991-1992, we studied 266 (8%, 22-23 strains monthly) of 3,337 Shigella isolates cultured during 2001-2002.

Transfer of ceftriaxone resistance plasmid by conjugation
Conjugation between ceftriaxone-resistant Shigella isolates (donor) and Escherichia coli K12 strain (F-, lac -, Nal R ) was performed according to the method of Neu et al. (20). Transconjugants were selected on MacConkey agar containing ceftriaxone (6 µg/mL) that produce lactose-fermenting pink colonies in contrast to non-lactose-fermenting pale colonies of Shigella. All putative transconjugants were examined for antimicrobial susceptibility and plasmid profiles to obtain transconjugants. They were tested for production of ESBL (18). All transconjugants were cultured twice on MacConkey agar plate containing ceftriaxone (6 µg/mL) to exclude contamination with donors. In the second transfer of R-plasmid by conjugation, ceftriaxone resistance and production of ESBL were transferred from transconjugants E. coli to ceftriaxone-susceptible and trimethoprim-sulphamethoxazole-resistant wild Shigella sonnei. The second-generation Shigella transconjugants were selected on MHA plates supplemented with ceftriaxone (6 µg/mL) and trimethoprim (24 µg/mL) and were tested for antimicrobial susceptibility, ESBL, and R-plasmid.

Plasmid analysis
Plasmid DNA was extracted from four ceftriaxone-resistant Shigella isolates and all transconjugants according to the method of Portnoy et al. (21) and was separated by electrophoresis in 0.5% agarose gel, stained, and visualized. Reference plasmid markers-V517 and 39R861were used for determining the size of unknown plasmids.

Statistical analysis
The significance of differences in the proportions of antimicrobial resistance and of the relative prevalence of each Shigella species was determined by the chi-square test. Two-tailed tests were applied.
Three (two S. sonnei and one S. boydii) of four TGC-resistant strains were susceptible to amoxicillin-clavulanate and positive in DDST, indicating production of a class A ESBL (19), as described earlier (22). We were able to transfer ß-lactam resistance and ESBL production of these TGCresistant strains to E. coli and Shigella by conjugation, which was encoded by a 50-MDa autotrasferable R-plasmid. The fourth isolate (S. flexneri) was resistant to amoxicillin-clavulanate but negative in DDST, thus exhibiting a class C (Amp C) ß-lactamase phenotype (19) mediated by a 94-MDa autotrasferable R-plasmid (22).

DISCUSSION
Shigella causes invasive infection of the intestine that presents the most pressing challenge for providing effective antimicrobial therapy. Due to the emergence of resistance, antimicrobial agents, such as sulphonamides, tetracycline, ampicillin, trimethoprim-sulphamethoxazole, nalidixic acid, and mecillinam have all in suc-  (2,7,(12)(13)(14)23). During the past several decades, the organisms have progressively become resistant to most useful and inexpensive antimicrobial agents (7,8,11). Our study demonstrates an increasing incidence and spectrum of antimicrobial resistance of Shigella isolates in Bangladesh in [2001][2002]. Rates of resistance to ampicillin, trimethoprim-sulphamethoxazole, and nalidixic acid increased to more than 50%. The resistance to tetracycline and chloramphenicol, which are not used currently for treating shigellosis, remained high and unchanged during the last decade. It is likely that the resistance rates ob-served in our hospital-based microbiology laboratory reflect the prevalence of resistance that exists in the community since nearly all cases of shigellosis were community-acquired, and the cultures were obtained on the day of admission. By analyzing the trends in the resistance patterns of various Shigella spp., we found that S. dysenteriae was at present significantly more resistant, followed by S. flexneri and other Shigella spp., in Bangladesh, especially to commonly-used antimicrobial agents. This finding is of special importance because S. flexneri is at present the predominant species in Bangladesh, like many other developing countries. At present, pivmecillinam (oral form of mecillinam), fluoroquinolones, azithromycin, and third-generation cephalosporins (cefixime) are used in many countries for treating shigellosis caused by Shigella resistant to all first-line drugs. Pivmecillinam is currently used as an empirical antimicrobial therapy for shigellosis in Bangladesh with caution since resistance to it is emerging. The rate of mecillinam resistance, observed in the present study, is significantly lower than that observed in our earlier study in Bangladesh (11). This was due to lack of precise guidelines for determining susceptibility to mecillinam in the past that inherently overestimated the resistance rate. We used the CLSI (formerly NC-CLS) methodology (24) for the determination of mecillinam susceptibility that reflects the true resistance rate in the present study. Azithromycin was found to be effective in treating shigellosis both in children and adults, including multidrug-resistant Shigella-associated infections (6,13). High modal MICs (6 µg/mL) and MIC 90 (8 µg/mL) of azithromycin for Shigella isolates were observed in our study, and we detected in-vitro resistance to azithromycin in Shigella for the first time in Bangladesh. It is not clear why Shigella exhibits high modal MIC values to azithromycin. It is surprising that a significant proportion of isolates are resistant to this drug, although the drug is not commonly used for treating shigellosis in Bangladesh. However, the frequent use of macrolides for other infections and high carriage rate of Shigella in the gut of apparently healthy humans in Bangladesh might contribute to the emergence and spread of azithromycin-resistant Shigella strains (25). The significance of in-vitro resistance of Shigella to azithromycin is still unknown since intracellular concentration of azithromycin achieved in colonic cells and leucocytes exceeds serum concentration by 100-fold or more that could be fatal for intracellular bacteria (26). It should be mentioned that the two zones of inhibition produced by azithromycin by the disc-diffusion method and E-test sometimes caused difficulty in interpretation of results.
Quinolones are a good choice for the treatment of shigellosis in adults (3,13). Nalidixic acid was effective (23) and approved for use in the treatment of shigellosis in children aged less than three months. However, >50% of our Shigella isolates were resistant to nalidixic acid like many other developing countries (2,3,6,8). Fluoroquinolones (ciprofloxacin and norfloxacin) are also effective in treating nalidixic acid-resistant shigellosis, but paediatric use is limited by concerns about arthopathy and chondrotoxicity. However, reported data suggest that they are generally safe for the treatment of shigellosis in children (3,27). In 1994, S. dysenteriae type 1, resistant to nalidixic acid having decreased susceptibility to ciprofloxacin (MIC >0.125 µg/mL), was reported for the first time in Bangladesh by our group (9). The isolation rate of such strains of Shigella remarkably increased to 71% in 2001-2002 involving all species. The importance of strains having decreased susceptibility to fluoroquinolones was revealed recently by an outbreak of S. dysenteriae type 1 in 2002 in eastern India that affected 1,728 persons (attack rate of 25.6%), resulting in 16 deaths (28). Suboptimal clinical responses, therapeutic and microbiologic (positive culture after therapy) failures were associated with decreased ciprofloxacin susceptibilities of clinical isolates of Salmonella spp. and other bacteria in many countries, including India, the UK, Denmark, and the USA (29)(30)(31). To reduce such risks for humans, a recommendation has recently been made to lower breakpoint to 0.125 µg/mL for fluoroquinolones for Salmonella (29). With the increasing prevalence of Shigella strains having decreased susceptibility to fluoroquinolones such as ciprofloxacin, there is a need for careful observation of the outcome of ciprofloxacin therapy for shigellosis to detect suboptimal clinical response or therapeutic failures, if any. However, the problem is that the strains having decreased susceptibility to ciprofloxacin are not reported as these appear susceptible when subjected to ciprofloxacin-susceptibility testing (disc-diffusion method or by current MIC breakpoints) by the CLSI guidelines. As suggested by our study, resistance to nalidixic acid appears to be a useful screening marker for decreased ciprofloxacin susceptibility. Hence, future studies should evaluate the clinical outcome of the treatment of shigellosis caused by strains having susceptibility to ciprofloxacin but resistance to nalidixic acid. Further, in our earlier study, we detected a single-point mutation at codon Ser83 (TGC) to Tyr83 (TTC) in the quinolone resistance-determining region of gyrA gene of S. dysenteriae type 1, resulting in resistance to nalidixic acid with decreased susceptibility to ciprofloxacin (9). Additional mutations in the same gene (codon 87) and/or parC (codons 80 and 84) are known to result in complete resistance to ciprofloxacin (15). Thus, the use of fluoroqui-nolones is likely to result in complete resistance in Shigella strains harbouring resistance to nalidixic acid by additional mutations that we have witnessed recently in Bangladesh and India (32)(33). Interesting is the fact that the recent isolates of ciprofloxacin-resistant S. dysenteriae type 1 in Bangladesh and India appeared to originate by such mechanisms: a mutation in codon 87 of the gyrA and additional mutation in codon 80 of parC genes (32)(33). Although R-plasmid-mediated quinolone resistance may occur on rare occasion (34), it is not unlikely that we will see more and more ciprofloxacin-resistant Shigellaassociated infections in the near future.
Cefixime and ceftriaxone were active against 98% of our isolates in vitro, but there is some dispute regarding the clinical efficacy of cefixime in treating shigellosis (6,35). Recently, cefixime was found to be clinically effective in 78% of children with shigellosis, predominantly caused by S. flexneri (6). However, it is an re-assuring finding that no increase in the MIC of ceftriaxone was observed in susceptible Shigella strains, unlike for ciprofloxacin or azithromycin. On the contrary, ESBL-mediated TGC resistance in Shigella strains was detected for the first time in Bangladesh. Detection of R-plasmid-mediated ESBL in Shigella isolates, transferable to E. coli K 12 and Shigella by conjugation, suggests that ESBL could spread resistance to third-generation cephalosporins among Shigella spp. and other pathogens in the community (22,(36)(37).
The limitation of our study is that we could not test all strains for antimicrobial susceptibility to detect exact rates of resistance to conventional and new useful antimicrobial agents. The isolates were from patients who came to the hospital for treatment or submitted faecal samples on the advice of physicians. Thus, strains might be associated with severe form of illnesses or from cases not responding to therapy reflecting high rates of resistance compared to those existing in the community. However, high rates of resistance among Shigella isolates have been reported in the community of Bangladesh (7,(11)(12).
Our study showed that Shigella strains developed resistance to many useful antimicrobial agents, including mecillinam, azithromycin, ceftriaxone, and cefixime in Bangladesh. Options for antimicrobial therapy for such MDR Shigellaassociated infections are very limited leaving fluo-roquinoloes as the only option. Detection of decreased susceptibility to fluoroquinoloes in a high proportion of Shigella strains and complete fluoroquinolones-resistant S. dysenteriae type 1 (15)(16)28,(31)(32) clearly demands careful and judicial use of these drugs to avoid rapid emergence and spread of resistance.
In conclusion, physicians should be aware of the high rates of antimicrobial resistance and increasing spectrum of resistance of Shigella spp. in Bangladesh. Continuous monitoring of the resistance patterns is essential, and antimicrobial susceptibility testing should be carried out on clinical isolates, and empirical antimicrobial therapy need to change accordingly. In addition, reduced susceptibility of Shigella strains to useful drugs should be identified by determination of MICs of antimicrobial agents for the early detection of the emergence of resistance. When indicated, pivmecillinam or ciprofoxacin might be considered for treating shigellosis with caution in Bangladesh.